Abstract:

A CDR grafted humanized rAb comprises a human Ig framework having CDRs
from murine mAb 1A4A1 VH and VL. DNA sequences and vectors incorporating
such sequences are also provided as are pharmaceutical preparations and
methods of using the humanized rAbs.

7. The humanized rAb of claim 1 having a VH comprising an amino acid
sequence according to SEQ ID NO: 7.

8. The humanized rAb of claim 1 having a VL comprising an amino acid
sequence according to SEQ ID NO: 8.

9. The use of the rAb of claim 1 for the treatment or prophylaxis of VEEV
infection.

10. A pharmaceutical preparation comprising as the active ingredient a
humanized rAb as claimed in claim 1 or a fragment thereof and a
pharmaceutically acceptable carrier or diluent.

11. A DNA sequence which encodes a polypeptide corresponding to a CDR
grafted VH having an amino acid sequence according to SEQ ID NO: 7.

12. A DNA sequence which encodes a polypeptide corresponding to a CDR
grafted VL having an amino acid sequence according to SEQ ID NO: 8.

13. A cloning or expression vector containing a DNA sequence which encodes
a polypeptide corresponding to a CDR grafted VH having an amino acid
sequence according to SEQ ID NO: 7 or a CDR grafted VL having an amino
acid sequence according to SEQ ID NO: 8.

14. A host cell transformed with a cloning or expression vector according
to claim 13.

15. A method of treatment or prophylaxis against VEEV infection in a
mammal comprising administering to said mammal the rAb according to claim
1.

16. The humanized rAb of claim 1 wherein said rAb has an amino acid
sequence according to SEQ ID NO:12 or SEQ ID NO:14.

[0002]Venezuelan equine encephalitis virus (VEEV), a member of the
alphavirus genus of the family Togaviridae, is an important
mosquito-borne pathogen in humans and equides [1]. VEEV infections mainly
target the central nervous system and lymphoid tissues causing severe
encephalitis in equines and a spectrum of human diseases ranging from
unapparent or sub-clinical infection to acute encephalitis. Neurological
disease appears in 4-14% of cases. The incidence of human infection
during equine epizootics could be up to 30%. Mortality associated with
the encephalitis in children is as high as 35%. Recent outbreaks in
Venezuela and Colombia in 1995 resulted in around 100,000 human cases
with more than 300 fatal encephalitis cases [2]. Furthermore, VEEV is
highly infectious by aerosol inhalation in humans and other animals.
However, there are no antiviral drugs available that are effective
against VEEV although currently there are two forms of IND
(investigational new drug) VEEV vaccines available for human and
veterinary use: TC-83, a live-attenuated Trinidad donkey strain and C-84,
a formalin-inactivated TC-83 [3,4]. However, for various reasons, these
vaccines are far from satisfactory. For example, approximately 20% of
recipients that receive the TC-83 vaccine fail to develop neutralizing
Abs, while another 20% exhibit reactogenicity. In addition, the TC-83
vaccine could revert to wild-type form. The vaccine C-84 is well
tolerated, but requires multiple immunizations, periodic boosts, and
fails to provide protection against aerosol challenge in some rodent
models.

[0003]Like the other alphaviruses, VEEV is an enveloped virus, consisting
of three structural proteins: a capsid encapsidating the viral RNA
genome, and two envelope glycoproteins, E1 and E2. E1 and E2 form
heterodimers, which project from the virus envelope as trimer spikes.
Epitopes on the spikes are the targets of neutralizing Abs. Studies have
shown that the viral neutralizing epitopes are mainly located on the E2
protein, and that the E2C epitope appears to be the hub of the
neutralization epitopes [5,6]. The murine monoclonal Ab (mAb) 1A1A4 [14]
is specific for E2C. This mAb has been shown to be efficient in
protecting animals from a lethal peripheral challenge with virulent VEEV
[7].

[0004]Murine mAbs, however, have serious disadvantages as therapeutic
agents in humans [8]. For example, one of the problems associated with
using murine mAbs in humans is that they may induce an anti-mouse Ab
response. Further, repeat administration of murine mAbs may result in
rapid clearance of the murine mAbs and anaphylaxis, which can sometimes
be fatal. To overcome this hurdle, the humanization of murine mAbs has
been proposed, by which process murine Ab frameworks are replaced by
human Ab ones in order to reduce immunogenicity of Abs in humans [9,10].

[0007]In one aspect, the invention provides a humanized rAb comprising a
human immunoglobulin (Ig) framework and having grafted thereon
complementarity determining regions (CDRs) from the murine mAb 1A4A1. In
a preferred embodiment, the human 1g framework is obtained from IgG1.

[0008]In another aspect, the invention provides a humanized rAb having
specificity to the E2 envelope protein of VEEV. More specifically, the
rAb has specificity to the E2c epitope of the E2 protein.

[0009]In another aspect, the invention provides a humanized rAb wherein
the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy
chain variable region (VH) have the following amino acid sequences:

[0010]In another aspect, the invention provides a humanized rAb wherein
the complementarity determining regions CDR1, CDR2 and CDR3 of the light
chain variable region (VL) have the following amino acid sequences:

[0018]In other aspects, the invention provides methods and uses for
treatment or prophylaxis of VEEV infection utilizing the rAbs described
herein. The invention also provides pharmaceutical preparations for such
treatment or prophylaxis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]These and other features of the invention will become more apparent
in the following detailed description in which reference is made to the
appended drawings wherein:

[0020]FIG. 1 is a representation of the external structure of the VEEV.

[0023]FIG. 4 schematically illustrates the cloning of the murine Ab VH and
VL.

[0024]FIG. 5 schematically illustrates the humanization of the Ab VH and
shows its amino acid sequence.

[0025]FIG. 6 schematically illustrates the humanization of the Ab VL and
shows its amino acid sequence.

[0026]FIG. 7 schematically illustrates the design of a full Hu1A4A1IgG1
rAb gene in a single open reading frame with two versions,
Hu1A4A1IgG1-furin and Hu1A4A1IgG1-2A.

[0027]FIG. 8 schematically illustrates the cloning of the
Hu1A4A1IgG1-furin and Hu1A4A1IgG1-2A genes into an adenoviral vector
respectively.

[0028]FIG. 9 schematically illustrates expression and purification of the
Hu1A4A1IgG1-furin and Hu1A4A1IgG1-2A rAbs.

[0029]FIGS. 10 and 11 illustrate the results from the SDS-PAGE separation
of the produced Hu1A4A1IgG1-furin rAb.

[0030]FIG. 12 illustrates the results from the sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of the
produced Hu1A4A1IgG1-2A rAb.

[0031]FIG. 13 illustrates the results of the enzyme-linked immunosorbent
assays (ELISA) for the reactivity of the Hu1A4A1IgG1-furin and
Hu1A4A1IgG1-2A rAbs.

[0032]FIG. 14 schematically illustrates Hu1A4A1IgG1-2A was cleaved between
the heavy and light chains as expected, whereas Hu1A4A1IgG-furin was not
cleaved.

[0033]FIG. 15 schematically illustrates the neutralization assay used in
assessing the neutralizing activity of the Hu1A4A1IgG1-furin and
Hu1A4A1IgG1-2A rAbs against VEEV.

DETAILED DESCRIPTION OF THE INVENTION

[0034]FIG. 1 illustrates the external structure of the VEEV. As shown, the
virus 10 includes a nucleocapsid 12 enveloping the viral RNA genome. The
envelope comprises glycoproteins E1 and E2, arranged in the form of
heterodimers 14. Protein E2, which is responsible for viral attachment to
the host cell, contains neutralizing epitopes.

[0035]As has been described in the prior art, the murine mAb 1A4A1 has
been found to be specific to the VEEV E2 envelope protein and, further,
has been found to have a strong neutralizing function against VEEV. The
murine mAb, however, causes a sometimes fatal allergenic reaction in
humans, resulting in the formation of human anti-mouse Abs (HAMA). It is
for this reason that the present inventors have sought to humanize the
1A4A1 mAb so as to provide an effective agent to counter VEEV infection
in humans.

[0036]In vivo efficacy studies in mice have demonstrated that treatment
with murine mAb 1A4A1 leads to protection of animals from a lethal
peripheral challenge with virulent VEEV. Thus, the present invention
builds upon these findings by providing a humanized mAb 1A4A1 to reduce
the foreignness of murine mAb in humans. For doing this, the majority of
the non-human protein sequence (in one embodiment, more than 90%) of mAb
1A4A1 is replaced with a human Ab sequence and the resultant whole
humanized mAb gene is then synthesized and cloned to an adenoviral
vector. The recombinant adenoviral vector can be delivered as a
therapeutic agent for prophylaxis or treatment of VEEV infection in
humans. One advantage of this method is that the vector can express the
humanized Ab in the human body for a long period of time. The humanized
Ab can also be produced in cell culture and delivered directly as a
therapeutic.

[0037]The humanization of the present anti-VEEV mAb 1A4A1 has not been
done previously and particularly not for the prophylaxis or treatment of
VEEV infection. The present invention provides in one embodiment a
humanized Ab, referred to herein as Hu1A4A1IgG1, that retains the
VEEV-binding specificity and neutralizing activity of murine 1A4A1 while
not eliciting a HAMA response. As described further below, the humanized
Ab comprises an Ig framework of human IgG1 and CDRs obtained from murine
mAb 1A4A1. The rAb of the present invention is specific to an epitope of
the E2 envelope glycoprotein of VEEV and, more specifically, to the
E2c epitope thereon.

[0038]The construction of the humanized Ab of the invention is
schematically illustrated in FIGS. 2a to 2d. FIG. 2a illustrates
schematically the structure of a murine Ab 16 containing murine CDRs 18
on the respective variable regions. FIG. 2b shows a human Ab 20
containing human CDRs 22. As shown in FIG. 2c, a chimeric Ab 26 would
comprise the murine variable regions 24, containing the murine CDRs 18,
joined to the constant regions of the human Ab. On the other hand, FIG.
2d illustrates a humanized Ab 28 according to an embodiment of the
invention, wherein only the murine CDRs 18 are grafted to the variable
regions of the human Ab 20.

[0039]The substitution of the murine CDRs into the human Ig framework is
illustrated also in FIGS. 3a to 3c. As shown, the humanized Ab variable
region comprises the grafted CDRs, 18, from the murine Ab.

[0040]The protein sequences of the rAbs of the invention include linker
sequences. The expressed rAbs of the invention have amino acid sequences
as shown in SEQ ID NO:12 and SEQ ID NO:14. The nucleic acid constructs
used in transfecting cells to express the above rAbs are shown in SEQ ID
NO:11 and SEQ ID NO:13.

EXAMPLES

[0041]The following examples are provided to illustrate embodiments of the
present invention. The examples are not intended to limit the scope of
the invention in any way.

[0045]Murine mAb 1A4A1 was provided by Dr. J. T. Roehrig (Division of
Vector-borne Infectious Diseases, Centers for Disease Control and
Prevention, Fort Colins, Colo., USA). The VH and VL of mAb 1A4A1 were
cloned in a single chain variable fragment (ScFv) format, mA116
previously [7], which showed to retain the same binding specificity as
mAb 1A4A1 [11]. The humanization of VH and VL of murine mAb 1A4A1 was
done by Absalus Inc. (Mountain View, Calif., USA). Briefly, in order to
select human VH and VL frameworks 1-3, the VH and VL amino acid sequences
of murine 1A4A1 were separately subjected to IgBlast and IMGT searches
against the entire human Ig germline V gene segments and then human heavy
and light chain germline V gene segments were selected based on their
highest CDR 1 and 2 similarities with those of murine 1A4A1 VH and VL
without consideration of framework similarity. Both human VH and VL
framework 4 were selected, respectively, from human heavy and light chain
J gene segments based on the highest similarities between human J gene
segments and murine 1A4A1 VH and VL CDR3. Finally, CDRs of murine 1A4A1
VH and VL were, respectively, grafted onto the frameworks of selected
germline V and J gene segments of human Ab heavy and light chains,
resulting in humanized 1A4A1 (Hu1A4A1). Furthermore, the Hu1A4A1 VH and
VL were, respectively, grafted onto human gamma 1 heavy chain CHs and
kappa 1 light chain CL to assemble the whole humanized Ab gene, resulting
in humanized 1A4A1IgG1 (Hu1A4A1IgG1). This process is illustrated in
FIGS. 3 to 6.

[0046]Construction, Expression and Purification of Hu1A4A1IgG1
(Hu1A4A1IgG1-furin and Hu1A4A1IgG1-2A)

[0047]The Hu1A4A1IgG1 DNA sequence (˜2 kb) is schematically
illustrated in FIG. 7. The nucleic acid sequence of the Hu1A4A1IgG1-furin
rAb is provided in SEQ ID NO:11 and the nucleic acid sequence of the
Hu1A4A1IgG1-2A rAb is provided in SEQ ID NO:13.

[0048]The Hu1A4A1IgG1 DNA sequences were synthesized as follows. As shown
in FIG. 7, a light chain leader sequence was provided upstream from the
light chain, followed by a furin or 2A linker (discussed further below)
before the heavy chain. The whole DNA sequence flanked by Kpn I and Hind
III was synthesized by GenScript Corporation (Scotch Plaines, N.J., USA)
and cloned into pUC57 vector, resulting in pUC57-Hu1A4A1IgG1-furin or
pUC57-Hu1A4A1IgG1-2A.

[0049]Recombinant adenovirus vectors expressing either Hu1A4A1IgG1-furin
or Hu1A4A1IgG1-2A were constructed using AdEasy® system (Qbiogene,
Carlsbad, Calif., USA) according to the manufacturer's protocol. Briefly,
the Kpn I-Hind III fragment of Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A was
ligated to a Kpn I-Hind III-digested pShuttle-CMV vector. The resulting
pShuttle construct was co-transformed with the pAdEasy-1 vector into
Escherichia coli BJ5183 cells to produce recombinant adenoviral genomic
constructs for Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A proteins. The
recombinant adenoviral constructs, pAd-Hu1A4A1IgG1-furin and
pAd-Hu1A4A1IgG1-2A were linearized with Pac I and transfected into HEK
293 cells (American Type Culture Collection, Manassas, Va., USA) cultured
in Dulbecco's Modified Eagle's Medium supplemented with 5% fetal bovine
serum (FBS) for amplification and then the amplified adenovirus was
purified by a chromatographic method. This procedure is illustrated in
FIG. 8.

[0050]As illustrated in FIG. 9, the expression of Hu1A4A1IgG1-furin or
Hu1A4A1IgG1-2A was achieved by first infecting HEK 293 cells with the
recombinant adenovirus pAd-Hu1A4A1IgG1-furin or pAd-Hu1A4A1IgG1-2A at a
multiplicity of infection (MOI) of 1. The infected cells were cultured
for one week and the culture supernatant was harvested. The expressed
Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A was purified using protein L agarose
gel from Pierce (Brockville, Ont., Canada). Briefly, culture supernatant
was dialyzed against phosphate buffer saline (PBS) (Sigma-Aldrich,
Oakville, Ont., Canada) for 12 h and then concentrated using PEG
(Sigma-Aldrich) to less than 50 ml. The concentrated sample was incubated
with 2 ml protein L agarose gel at 4° C. for 1 h. The gel and
supernatant mixture was then loaded to an empty column, which was
subsequently washed with binding buffer. Bound Hu1A4A1IgG1-furin or
Hu1A4A1IgG1-2A was eluted with elution buffer. The eluted Ab was further
desalted using an excellulose column (Pierce) and then concentrated by a
Centracon® YM-30 (Millipore Corp., Bedford, Mass., USA).

[0051]The amino acid sequence of the expressed Hu1A4A1IgG1-furin is shown
in SEQ ID NO:12 and the amino acid sequence of the expressed
Hu1A4A1IgG1-2A is shown in SEQ ID NO:14.

[0052]SDS-PAGE

[0053]Abs were separated by 10% SDS-PAGE gels using a Mini-PROTEAN® II
apparatus (Bio-Rad Laboratories, Mississauga, Ont., Canada). The bands
were visualized by SimplyBlue® safestain staining (Invitrogen,
Burlington, Ont., Canada). The molecular weights of the samples were
estimated by comparison to the relative mobility values of standards of
known molecular weights. The SDS-PAGE analyses of the purified
Hu1A4A1IgG1-furin are illustrated in FIGS. 10 and 11. FIG. 12 illustrates
the SDS-PAGE analysis of the purified Hu1A4A1IgG1-2A. As shown, lanes 1
and 3 correspond to purified Hu1A4A1IgG1 and control human IgG1 in a
non-reducing condition and lanes 2 and 4 correspond to purified
Hu1A4A1IgG1 and control human IgG1 in a reducing condition.

[0054]ELISA

[0055]The reactivity of purified Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A to
VEEV E2 antigen was determined by ELISA. Nunc Maxisorp® flat bottomed
96-well plates (Canadian Life Technologies, Burlington, Ont., Canada)
were coated overnight at 4° C. with recombinant VEEV E2 antigen at
a concentration of 10 μg/ml in carbonate bicarbonate buffer, pH 9.6.
The plates were washed five times with PBS containing 0.1% Tween®-20
(PBST) and then blocked in 2% bovine serum albumin for 2 h at room
temperature. After five washes with PBST, the plates were incubated for 2
h at room temperature with various concentrations of Hu1A4A1IgG1-furin,
Hu1A4A1IgG1-2A or 1A4A1 Abs diluted in PBST. Following five washes with
PBST, the plates were incubated for 2 h at room temperature with
horseradish peroxidase (HRP)-conjugated rabbit anti-human IgG fragment
crystallizable portion or HRP-conjugated rabbit anti-mouse IgG (Jackson
ImmunoResearch Laboratories Inc., West Grove, Pa., USA) diluted 1:5000 in
PBST. Finally, the plates were washed five times with PBST and developed
for 10 min at room temperature with a 3,3',5,5'-tetramethylbenzidine
substrate (Kirkegaard and Perry Laboratories). The reactions were read at
an absorbance of 650 nm by a microplate autoreader (Molecular Devices,
Sunnyvale, Calif., USA). The results of the ELISA Hu1A4A1IgG1-antigen
binding assay are illustrated in FIG. 13.

[0056]Neutralization Assay in Vitro

[0057]Neutralizing activity of each of Hu1A4A1IgG1-furin and
Hu1A4A1IgG1-2A against VEEV (strain TC-83) was analyzed by a plague
reduction assay. Briefly, each Ab was serially two-fold diluted and mixed
with an equal volume containing 50 plaque-forming units of virus per 100
μl. After mixtures were incubated for 1 h at room temperature, 200
μl of the mixture was inoculated in duplicate into wells of six-well
plates containing confluent Vero cell monolayers and incubated at
37° C. for 1 h. At the end of the incubation, the virus/Ab
mixtures were removed from the wells before the wells were overlaid by
tragacanth gum and then incubated for 2 days. The wells were stained with
0.3% crystal violet and plaques were counted. Neutralization titre was
expressed as the highest Ab dilution that inhibited 50% of virus plaques.
This procedure is illustrated in FIG. 15.

[0058]Results and Discussion

[0059]Different approaches have been developed to humanize murine Abs in
order to reduce the antigenicity of murine Abs in humans [9,10]. One
widely used approach is CDR-grafting, which involves the grafting of all
murine CDRs onto a human Ab frameworks. The human Ab frameworks are
chosen based on their similarities to the frameworks of the murine Ab to
be humanized. The CDR-grafting approach has been proven successful in
some cases. However, in many more instances, this humanization process
could result in CDR conformation changes, which affect the
antigen-binding affinity. To restore the affinity, additional work for
back-mutation of several murine framework amino acids, which are deemed
to be critical for CDR loop conformation, have to be done.

[0060]Recently, Hwang et al. [12] employed an approach which consisted of
grafting CDRs onto human germline Ab frameworks based on the CDR sequence
similarities between the murine and human Abs while basically ignoring
the frameworks. Because the selection of the human frameworks is driven
by the sequence of the CDRs, this strategy minimizes the differences
between the murine and human CDRs. This approach has the potential to
generate humanized Abs that retain their binding affinity to their
cognate antigen. Further, since all residues in frameworks are from human
Ab germline sequences, the potential immunogenicity of non-human Abs is
highly reduced.

[0061]Using the above approach, and as disclosed herein, the present
inventors humanized an anti-VEEV murine mAb 1A4A1. The amino acid
sequences of VH and VL from murine 1A4A1 were first aligned with human Ig
germline V and J genes. As shown in FIG. 5, the human heavy chain V gene
segment H5-51 and J gene segment JH4 were selected to provide the
frameworks for the murine 1A4A1 VH. Similarly, as shown in FIG. 6, for
the murine 1A4A1 VL, the human light chain V gene segment L15 and J gene
segment Jk3 were selected.

[0062]The identities of the CDR1 and CDR2 amino acid sequences between
murine 1A4A1 VH and the human H5-51 gene segment were 20% and 47%,
respectively, while the identity of the CDR3 between murine 1A4A1 VH and
the JH4 gene segment was 33%. For the light chain, the identities of the
CDR1 and CDR2 between murine 1A4A1 VL and the human L15 gene segment were
27% and 14%, respectively, while the identity of the CDR3 between murine
1A4A1 VL and human Jk3 gene segment was 22%. The CDRs of murine 1A4A1 VH
were then grafted onto the frameworks of selected human Ig germline H5-51
and JH4 gene segments, while the CDRs of murine 1A4A1 VL were grafted
onto human L15 and Jk3 gene segments. The hu1A4A1 VH was further grafted
onto the human gamma 1 heavy chain CHs to form a complete heavy chain,
while the VL was grafted onto the human kappa 1 light chain CL to form a
whole humanized light chain. This procedure is schematically illustrated
in FIGS. 5 and 6 with the end structure being illustrated in FIG. 7.

[0067]In order to express heavy and light chains in a monocistronic
construct, a six-residue peptide, RGRKRR (SEQ ID NO: 9) containing the
recognition site for the protease furin, designated as "furin linker", or
a twenty-four-residue peptide of the foot-and-mouth-disease virus
(FMDV)-derived 2A self-processing sequence, APVKQTLNFDLLKLAGDVESNPGP (SEQ
ID NO: 10), designated as "2A linker", was incorporated between the two
chains. The location of the furin or 2A linker within the nucleic acid
constructs of the Abs is illustrated in FIG. 7. Furin is a ubiquitous
subtilisin-like proprotein convertase, which is the major processing
enzyme of the secretory pathway [13]. The furin minimal cleavage site is
R-X-X-R; however, the enzyme prefers the site R-X-(K/R)-R. An additional
R at the P6 position appears to enhance cleavage. The FMDV-derived 2A
linker is able to cleave at its own C terminus between the last two
residues through an enzyme-independent but undefined mechanism, probably
by ribosomal skip, during protein translation. To get the expressed Ab to
be secreted to culture media, a leader sequence was added upstream to the
Ab gene. FIG. 7 illustrates the synthesized DNA sequence, of
approximately 2 kb, including the human Ab kappa light chain L15 leader
sequence, the humanized light chain (VL+CL), the furin or 2A linker, and
the humanized heavy chain (VH+CH1+CH2+CH3). This sequence was then cloned
into an adenoviral vector. The unique restriction sites, as also shown in
FIG. 7, flanking the V regions, which allow for efficient V region
replacement and at the heavy chain V-C region junction for generation of
fragment antigen-binding portion of Ab (Fab), were also designed.

[0068]Protein G and A columns are widely used for a quick purification for
Abs because of protein G and A binding to the Fc portion of Ig. However,
protein G and A cannot only bind to human Ig, but also bind to bovine Ig,
therefore they cannot be used for purification of Hu1A4A1IgG1-furin or
Hu1A4A1IgG1-2A in our study since pAd-Hu1A4A1IgG1-furin or
pAd-Hu1A4A1IgG1-2A-infected HEK 293 cells were cultured in the medium
with 5% FBS containing a high percentage of bovine Ig. Unlike protein G
and A, protein L binds Ig through interactions with the light chains.
Protein L only binds to Ig containing light chains of type kappa 1, 3 and
4 in human and kappa 1 in mouse. Most importantly, protein L does not
bind to bovine Ig. Since our humanized Ab has human kappa 1 chain, we
chose a protein L column to purify Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A to
eliminate co-purification of bovine Ig. In this way, the purity of
Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A was relatively high in SDS-PAGE as
shown in FIGS. 10, 11 and 12.

[0069]When the purified product was subjected to 10% SDS-PAGE,
Hu1A4A1IgG1-furin and Hu1A4A1IgG1-2 showed up in a different way. As
illustrated in FIG. 12, Hu1A4A1IgG1-2A showed the same patterns as a
control human IgG1, one band of ˜150 kDa in non-reducing condition
(intact disulfide bridges) and two bands, 50 kDa for heavy chains and 25
kDa for light chains (broken disulfide bridges) in reducing condition,
indicating that the 2A linker underwent self-processing perfectly. On the
other hand, Hu1A4A1IgG1-furin showed only one clear band of ˜75 kDa
in reducing condition observed as illustrated in FIGS. 10 and 11,
indicating that the furin linker was not cleaved. However, in another
study (data not shown), the same furin linker sequence was cleaved in
another Fab construct expressed in a mammalian system. This indicated the
conformation of expressed Hu1A4A1IgG1-furin probably rendered the furin
linker inaccessible to furin or that the sequence surrounding the furin
linker influenced furin cleavage.

[0070]The specific binding reactivities of purified Hu1A4A1IgG1-furin and
Hu1A4A1IgG1-2A to VEEV E2 antigen were examined by ELISA. As illustrated
in FIG. 13, both versions of the Hu1A4A1IgG1 were found to bind to VEEV
E2 in a dose-dependent manner, similar to the binding to VEEV E2 of its
parental murine 1A4A1, indicating this non-cleaved Ab was still reactive
to VEEV E2 antigen in ELISA. Furthermore, both versions were evaluated
for their ability to block VEEV infection in Vero cells using a standard
plaque-reduction assay. The Hu1A4A1IgG1-furin showed a neutralizing
activity with 50% plaque reduction neutralization titer at 0.78 μg/ml,
whereas Hu1A4A1IgG1-2A showed a much higher neutralization titre at 0.1
μg/ml.

[0071]From the above results, it is concluded that the murine 1A4A1 Ab was
successfully humanized. As illustrated in FIG. 14, the expressed and
purified Ab of Hu1A4A1IgG1-2A was cleaved between the heavy and light
chains as expected; however, Hu1A4A1IgG1-furin was not cleaved.
Nevertheless, the present inventors have exhibited that both versions of
the Hu1A4A1IgG1 retained the antigen binding specificity and virus
neutralizing activity. Thus, the Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A
discussed and characterized herein would serve as an effective
prophylactic and therapeutic agent against VEEV infection.

Example 2

In vivo Study--Protection of Mice from VEEV Challenge by Passive
Immunization with Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A

[0079]To determine the half-life of Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A in
mouse serum, groups of 4 mice, were injected i.p. with 50 μg, each
mouse, of either Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A, or human anti-VEEV
IgG and bled from the vein at increasing time intervals after injection.
The quantity of Ab present in serum samples was estimated by immunoassay.
Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A had a similar half-life as human
anti-VEEV IgG, around 10 days.

[0080]Protection of Mice from VEEV Challenge by Passive Immunization with
Hu1A4A1IgG1-Furin or Hu1A4A1IgG1-2A

[0081]Groups of 8 mice were injected i.p. with the Hu1A4A1IgG1-furin,
Hu1A4A1IgG1-2A, human anti-VEEV IgG or PBS alone and 24 h later
challenged s.c. with 100×LD50 of VEEV. None of the PBS alone
treated mice survived. All the Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A
treated mice survived the VEEV challenge without any clinical signs at 14
days post-challenge.

[0082]Discussion

[0083]Passive immunization of the Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A in
mice (50 pg/mouse) 24 h before virulent VEEV challenge provided 100%
protection against 100×LD50 challenge of VEEV when mice were
treated with 50 μg/each mouse of Hu1A4A1IgG1-furin or Hu1A4A1IgG1-2A.
The mice were also found to be asymptomatic throughout the 14 day
observation period. These results indicate that the humanized anti-VEEV
rAbs of the present invention has prophylactic capacity against VEEV
infections. The half-lives of the humanized anti-VEEV rAbs in mice was
around 10 days suggesting that the humanized anti-VEEV rAbs of the
invention would be an effective prophylactic against VEEV for at least
several weeks.

[0084]Bibliography

[0085]One or more of the following documents have been referred to in the
present disclosure. The following documents are incorporated herein by
reference in their entirety.

[0103]Although the invention has been described with reference to certain
specific embodiments, various modifications thereof will be apparent to
those skilled in the art without departing from the purpose and scope of
the invention as outlined in the claims appended hereto. Any examples
provided herein are included solely for the purpose of illustrating the
invention and are not intended to limit the invention in any way. Any
drawings provided herein are solely for the purpose of illustrating
various aspects of the invention and are not intended to be drawn to
scale or to limit the invention in any way. The disclosures of all prior
art recited herein are incorporated herein by reference in their
entirety.